Larry Ruben

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Pathways Involved in Environmental Sensing in Trypanosomatids

Digenetic parasites, such as those of the order Kinetoplastida, must respond to extracellular and intracellular signals as they adapt to new environments within their different hosts. Evidence for signal transduction has been obtained for Trypanosoma brucei, T. cruzi and Leishmania, as reviewed here by Marilyn Parsons and Larry Ruben. Although the broad picture suggests similarities with the mammalian host, there are large gaps in our understanding of these processes; this probably contributes to a perception of differences. Nonetheless, current evidence suggests that the trypanosomatids might lack certain classes of signalling molecules found in other organisms.

The RACK1 Homologue from Trypanosoma brucei Is Required for the Onset and Progression of Cytokinesis

The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.

Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24- and 38-kilodalton proteins.

The present study was undertaken to systematically purify calcium binding proteins (CaBPs) from homogenates of Trypanosoma brucei. This work is important since CaBPs either serve as intracellular calcium buffers or mediate cellular response to calcium signals. Disruption of either process should be lethal to trypanosomes. We report that the 45Ca-gel overlay assay can be used to detect CaBPs following fractionation on DE-52, phenyl-Sepharose, Mono-Q, and Superose 12. Specific CaBPs of 22, 24, and 38 kDa were purified. Each of these proteins associated with 45Ca under denaturing and non-denaturing conditions. An approximate Kd for calcium of 8 microM was calculated for 22-kDa CaBP. None of the trypanosome CaBPs were related to known calcium binding protein families. They did not associate with hydrophobic interaction columns or cellular membranes in a calcium-dependent way, nor cross-react with 2 separate antibodies against annexin consensus sequences. A synthetic peptide corresponding to amino terminal residues 16-30 of 22-kDa CaBP was used to generate polyclonal antibodies. Immunoblots identified 22-kDa CaBP in African trypanosomes but not in other Kinetoplastidae or mammalian cells. Nonetheless, significant homology (58%) was observed between the amino terminal 37 residues of 22-kDa CaBP and the amino terminus of translationally controlled p21 from mammalian tumor cells. The present study is the first to apply systemic fractionation techniques to identify the complement of CaBPs in T. brucei. We conclude that novel CaBPs other than calmodulin and annexin family members contribute towards calcium pathways in these organisms.

The human Aurora kinase inhibitor danusertib is a lead compound for anti-trypanosomal drug discovery via target repurposing

New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet drug discovery efforts are not often focused on this area due to cost. Target repurposing, achieved by the matching of essential parasite enzymes to those human enzymes that have been successfully inhibited by small molecule drugs, provides an attractive means by which new drug optimization programs can be pragmatically initiated. In this report we describe our results in repurposing an established class of human Aurora kinase inhibitors, typified by danusertib (1), which we have observed to be an inhibitor of trypanosomal Aurora kinase 1 (TbAUK1) and effective in parasite killing in vitro. Informed by homology modeling and docking, a series of analogs of 1 were prepared that explored the scope of the chemotype and provided a nearly 25-fold improvement in cellular selectivity for parasite cells over human cells.

The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms

Aurora kinase family members co‐ordinate a range of events associated with mitosis and cytokinesis. Anti‐cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase‐1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti‐parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl‐tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC50 of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC50 of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi‐dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high‐affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small‐molecule inhibitors.

Selective Transfer of Calcium from an Acidic Compartment to the Mitochondrion of Trypanosoma brucei MEASUREMENTS WITH TARGETED AEQUORINS

Organelle compartments are used by cells as reservoirs of exchangeable Ca2+ and as Ca2+ buffers. The following study uses recombinant aequorins (CYT-AEQ and MT-AEQ) to measure the dynamics of Ca2+ flux between organelles in procyclic forms of the pathogenic protozoan, Trypanosoma brucei. Emphasis is placed on the exchange between an acidic Ca2+ reservoir and the mitochondrion. The mammalian mitochondrial targeting sequence was functional in trypanosomes as determined by immunoblots, immunolocalizations, and the observation that MT-AEQ was in a compartment whose Ca2+ uptake was inhibited 82% with carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN. The resting level of free calcium ion concentration in the mitochondrion ([Ca2+]mit) was slightly higher than that in the cytoplasm ([Ca2+]cyt) (400 ± 50 nm and 290 ± 40 nm, respectively). Melittin (125 nm) disrupted Ca2+homeostasis by inducing Ca2+ influx across the plasma membrane. [Ca2+]cyt became slightly elevated to 410 ± 100 nm, whereas [Ca2+]mit was selectively increased approximately 12-fold, with a broad peak at 4.8 ± 1.9 μm. At the peak, the mitochondrion contained approximately three times more free Ca2+ than the cytosol. However, mitochondrial retention of the Ca2+ was transient. Similar selective transport into the mitochondrion was observed when Ca2+ efflux from an acidic compartment was induced with monensin (2 μg/ml) in the presence of 5 mm EGTA. [Ca2+]cyt was transiently elevated to 400 ± 50 nm, whereas [Ca2+]mit was elevated to 3.3±1.3 μm. When cells were treated sequentially with monensin (2 μg/ml) and then melittin (200 nm), mitochondrial Ca2+ transport was normal. However, [Ca2+]cyt became elevated to a level that was 1.4-fold higher than with melittin alone. Overall, these data demonstrate that the trypanosome mitochondrion is not a reservoir of exchangeable Ca2+ in the resting cell. However, Ca2+ is selectively channeled to the mitochondrion from the plasma membrane or acidic Ca2+ storage compartment. Additionally, the acidic compartment contributes to maintenance of Ca2+ homeostasis in response to melittin.

The RACK1 Signal Anchor Protein from Trypanosoma brucei Associates with Eukaryotic Elongation Factor 1A: A Role for Translational Control in Cytokinesis

RACK1 is a WD‐repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo‐EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor‐1a (eEF1A) as determined by tandem MS of TAP‐TbRACK1 affinity eluates, co‐sedimentation in a sucrose gradient, and co‐precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow.

Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids

Previously, we showed that arachidonic acid (AA) stimulates Ca2+ currents in pathogenic Trypanosoma brucei (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid. Biochem. J 1998;336:659-666). Here we examine the mechanism used by T. brucei to release AA from the sn-2 position of diacyl glycero-phospholipids. We report that T. brucei accomplishes this feat in the apparent absence of phospholipase A2 (PLA2). Instead, deacylation is initiated at the sn-1 position, followed by acyl migration and hydrolysis with LysoPLA. Neither whole cell homogenates nor enriched protein fractions could release AA from substrates whose sn-1 position contained a non-hydrolyzable alkyl ether linkage. These same fractions however, released AA from ester linked phospholipids, and TLC analysis of the reaction products supported the sequential deacylation process. The release of sn-2 AA from 1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-PC was linear up to 90 min at an average rate of 50 nmol x min(-1) x mg(-1). sn-2 AA was processed more efficiently than sn-2 palmitate. The reaction was also greatest for: LysoPC>diacyl-PC (sn-1 labeled)>diacyl-PC (sn-2 labeled). Product formation was sensitive to polar head group, and PI was processed at less than 10% the rate of PC or PE. The enzymatic deacylation was inhibited by the serine specific reagent, methyl arachidonyl fluorophosphonate (MAFP) and the cysteine reagent N-ethylmaleimide (NEM). Both NEM and MAFP inhibited LysoPLA activity under conditions where there was little effect on PLA1 activity. Overall, we conclude that T. brucei can release AA from diacyl glycero-phospholipids by a sequential deacylation process. Two independent active sites appear to be involved. Interestingly, a high percentage of inner leaflet phospholipids are protected from degradation since they occur in the non-hydrolyzable 1-alkyl ether form.

Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines

The following study was undertaken to determine whether an inducible calcium influx pathway is present in intact bloodstream forms of Trypanosoma brucei. Fura-2 fluorescence was used to demonstrate that amphiphilic peptides and amines, including melittin, mastoparan and compound 48/80, each produced a dose dependent calcium influx across the plasma membrane. Calcium influx did not result from general disruption of membrane integrity, since a corresponding influx of ethidium bromide or other divalent cations was not observed. Instead, the calcium influx was selectively blocked by the calcium channel antagonists, La3+, Cd2+ or Ni2+, and was not affected by the Na+ channel antagonists, tetrodotoxin or amiloride. Activation of the trypanosome calcium influx pathway was dependent upon an intact membrane potential, and the rise in intracellular free calcium concentration ([Ca2+]i) was reversed upon membrane depolarization with gramicidin D. Changes in Ins(1,4,5)P3 did not accompany the calcium influx. Overall, these data provide the first evidence of an inducible calcium influx pathway in T. brucei, and describe methods to selectively manipulate this pathway.